chronopharmacology of melatonin in mice to maximize the...

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Chronopharmacology of melatonin in mice to maximize the antitumor effect

and minimize the rhythm disturbance effect

Takanori Akagi1, Kazami Ushinohama1, Shoko Ikesue1, Eiji Yukawa1, Shun Higuchi1,

Kenji Hamase2, Kiyoshi Zaitsu2, Shigehiro Ohdo1

1 Department of Clinical Pharmacokinetics, 2: Department of Bio-Analytical

Chemistry Division of Pharmaceutical Science, Graduate School, Kyushu University,

3-1-1, Maidashi, Higashi-Ku, Fukuoka, 812-8582 Japan.

(T.A., K.U., S.I., E.Y., S.H., K.H., K.Z., S.O.)

Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.

JPET Fast Forward. Published on October 16, 2003 as DOI:10.1124/jpet.103.055657This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on October 16, 2003 as DOI: 10.1124/jpet.103.055657

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Running title;

melatonin receptor expression and pharmacological effect

Correspondence author;

Shigehiro Ohdo, Ph.D.

Department of Clinical Pharmacokinetics, Division of Pharmaceutical Science,

Graduate School, Kyushu University, 3-1-1, Maidashi, Higashi-Ku, Fukuoka,

812-8582 Japan.

Phone:092-642-6658,

Fax:092-642-6660,

E-mail:[email protected]

Document statistics;

Text Pages; 25 pages

Tables; 3 items

Figures; 6 items

References; 40 items

Abstract; 218 words

Introduction; 595 words

Discussion; 1,303 words

Abbreviations;

MLT, melatonin;

SCN, suprachiasmatic nucleus;

S-180, sarcoma 180;

AUC, area under the concentration-time curve;

MRT, mean residence time

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on October 16, 2003 as DOI: 10.1124/jpet.103.055657

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Abstract

The influence of dosing time on the antitumor effect and the rhythm disturbance

effect of melatonin (MLT) was investigated in ICR male mice under a light-dark

(12:12) cycle. In tumor-bearing mice, the antitumor effect of MLT (1 mg/kg,

intraperitoneal) was most effective in the dark phase; and the rhythm disturbance

effect of MLT on the locomotor activity was more serious in the light phase than in the

dark phase. The antitumor effect and the rhythm disturbance effect of MLT increased

when the specific binding of MLT receptor in target tissues, tumor or suprachiasmatic

nucleus (SCN), increased and they decreased when the level decreased.

Furthermore, since luzindole (LZD), a both MT1 and MT2 blocker, caused the

antitumor effect or rhythm disturbance effect of MLT to decrease, it is suggested that

the time-dependent change of the pharmacological effects of MLT were influenced by

that of MLT receptor(s) function. On the other hand, there was no significant dosing

time-dependent change of MLT concentration in tumor or brain after injection. Thus,

the pharmacokinetic factor does not appear to contribute to the dosing


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time-dependent effect of MLT. These results suggest that by choosing the most

suitable dosing time for MLT, the efficacy of the drug can be increased and the toxicity

of the drug can be decreased in certain experimental and clinical situations.


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A large number of rhythmic variables are influenced by environmental factors such

as light, temperature, and social communication that vary cyclically in nature and

serve to synchronize biological rhythms to the daily rotation of the earth. Responses

to a variety of drugs show 24-hr rhythmicity (Ohdo et al., 1991, 1996, 1998; Watanabe

et al., 1992). Use of a chronopharmacological strategy can improve the outcome of

pharmacotherapy. However, several drugs cause alterations in the 24-hr rhythms of

biological, physiological and behavioral processes (Duncan, 1996; Horikawa et al.,

2000; Ohdo et al., 2001). The alteration of rhythmicity is sometimes associated with

therapeutic effects, or may lead to illness and altered homeostatic regulation.

MLT is an indoleamine synthesized from serotonin in the pineal gland. MLT has

many pharmacological and physiological effects. One of them is the antitumor effect.

Both in vitro and in vivo, MLT has been found to inhibit neoplastic growth and to delay

tumor progression (Cini et al., 1998). Moreover, MLT is well known to show no toxic

effects (Lissoni et al., 1993a, 1993b), and, therefore, as a non-toxic anticancer agent.

In phase II-type clinical trials, the effectiveness of MLT was shown either alone or in

combination with other chemotherapeutic drugs, as an anticancer agent (Panzer and


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Viljoen, 1997). Although very little is known about the exact mechanisms by which

MLT inhibits tumor growth, it has been shown that MLT has both a direct oncostatic

effect on tumor cells (Blask, 1993, 1997) and an anticancer action via stimulation of

the immune system (Conti and Maestroni, 1995). It was suggested that both direct

and indirect antitumor effects are via its specific binding site in tumor cells or immune

cells. Some drugs, which elicit their actions via their specific binding sites, have been

shown to have a dosing time-dependent effect in relation to their receptor sensitivity

(Takane et al., 2000). Therefore, it is probable that the antitumor effect of MLT

changes according to the time of administration. However, the influence of MLT

dosing time on tumor growth has not been revealed.

MLT has an effect on biological rhythms similar to light. The effects of MLT

treatment or light exposure on biological rhythms follow a phase-response curve.

Light exposure in the late dark phase causes a phase advance in the sleep-wake

cycle, while light exposure in the early dark phase causes a phase delay. However,

MLT treatment produces the opposite effects on biological rhythms to light (Lewy et

al., 1992). Namely, MLT treatment in the late light phase causes a phase advance in


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the sleep-wake cycle, while MLT treatment in the early light phase causes a phase

delay. This effect of MLT appears to act via the SCN, the principal circadian clock.

Due to this effect, in clinical situations, MLT is used to cure sleep disorders, jet lag

and so on. However, a low dose of MLT (0.1-10 mg) given in the middle of light phase

when endogenous levels are lowest causes decreased sleep-onset latency,

decreased oral temperature and a reduction in the number of correct responses on

the Wilkinson auditory vigilance task (Dollins et al., 1993). Therefore, if dosing time is

not appropriate, this effect of MLT may cause rhythm disorders.

The purpose of this study was to investigate the influence of MLT dosing time on

tumor growth in tumor-bearing mice to maximize the antitumor effect and to

investigate the influence of MLT dosing time on the locomotor activity to minimize the

risk of rhythm disturbance caused by exogenous MLT. The mechanism underlying the

dosing time-dependent difference was elucidated from the viewpoints of MLT

pharmacodynamics or pharmacokinetics.


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Methods

Animals and cell culture. Four-week-old male ICR mice (Charles River Japan, Inc.,

Kanagawa, Japan) were housed 3-10 per cage under standardized light-dark cycle

conditions (lights on at 07:00 off at 19:00) at a room temperature of 24 ± 1°C and

humidity of 60 ± 10% with food and water ad libitum. All mice were adapted to their

light-dark cycle for 2 weeks prior to experiments. Sarcoma 180 (S-180) cells

(Dainippon Pharmaceutical Co. Ltd., Osaka, Japan), were grown in eagle’s minimal

essential medium supplemented with 10% FBS, 0.05% kanamycin, penicillin and

streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. S-180 cells

were transplanted with 5x105 cells into the hind footpads of mice at the day before

drug injection.

Preparation of dosing solutions. MLT (Wako Pure Chemical Industries Ltd.,

Osaka, Japan) dissolved in saline to yield an appropriate concentration of 0.1 mg/ml.

Luzindole (LZD, 2-benzyl-N-acetyltryptamine) (Wako Pure Chemical Industries Ltd.,

Osaka, Japan) was prepared in 50% ethanol and then diluted in Krebs' solution to


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appropriate concentrations. The final ethanol concentrations in the tissue bath did not

affect contractility. MLT and LZD were used at an intraperitoneal (ip) dose of 1 mg/kg

or 10 mg/kg by means of 23-gauge needle connected to a 1-ml syringe. The volume

of drug solutions administered was 10 ml/kg. Other reagents, purchased from Wako

Pure Chemical Industries Ltd. (Osaka, Japan), were of analytical grade and used

without further purification.

Influence of MLT dosing time on growth of tumor weight. Firstly, groups of 9-10

mice were intraperitoneally injected with MLT (1 mg/kg) at 07:00, 13:00, 19:00 or

01:00 to investigate the influence of dosing time on the antitumor effect of MLT.

Secondly, we selected two different dosing times such as peak and trough. Groups of

9-10 mice were intraperitoneally injected with MLT (1 mg/kg), MLT and LZD (10

mg/kg) or saline at 13:00 or 01:00. Drug or saline was administered daily for 8 days.

On the 8th day, the growth of S-180 cells was monitored by calculating the tumor

volume from caliper measurements. The tumor weight was calculated according to

the following equation: Tumor weight = AxB2/2. where A is the longer diameter and B

is the shorter one (mm).


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Influence of MLT dosing time on locomotor activity. After groups of 3 mice were

intraperitoneally injected with saline for 5 days, they were intraperitoneally injected

with MLT (1 mg/kg), MLT and LZD (10 mg/kg) or saline at 13:00 or 01:00. Drugs were

administered daily for 7 days. Locomotor activity was measured using scanet

SV-10LD (Toyo Sangyo Co. Ltd., Toyama, Japan), and the activity count (number of

movements) was recorded at 1 hr intervals. For visualization of locomotor activity

rhythm, hourly the activity counts were calculated using a moving average with a 4-hr

window. The average counts were plotted at 09:00, 13:00, 17:00, 21:00, 01:00 or

05:00.

Time-dependent change in specific binding of MLT to tumor or SCN cells.

Tumor tissues were removed from 6 mice at 07:00, 13:00, 19:00 or 01:00. To obtain

the three SCN samples, coronal brain slice were prepared from 18 mice using rodent

brain matrix (RBM-2000C; ASI Instruments Inc., Warren, Michigan, USA) at 13:00 or

01:00. Tumor or SCN was homogenized in 1 ml of ice-cold 50 mM Tris-HCl buffer (pH

7.4). Homogenates were then centrifuged at 15,000 rpm for 15 min at 4°C. The

obtained pellet was resuspended in 4 or 1 (tumor 4, SCN 1) ml of Tris-HCl buffer (pH


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7.4). After that, homogenates were centrifuged again. Pellets were resuspended in 3

or 1 (tumor 3, SCN 1) ml of Tris-HCl buffer. The protein concentration was

approximately 3 or 1 (tumor 3, SCN 1) mg/ml using Lowry's method (DC Protein

Assay; Bio-Rad, Hercules, CA, USA). The binding assay was done in the method

already described (Takane et al., 2000). The binding assay was performed with a

reaction mixture (total volume, 200 µl) containing 100 µl of aliquot of tumor

homogenate and 50 nM [O-methyl-3H] MLT (Amersham Pharmacia Biotech Ltd, Little

Chalfont, Buckinghamshire, UK) or containing 100 µl of aliquot of SCN homogenate

and 12.5 to 50 nM [O-methyl-3H] MLT. Non-specific binding was determined in the

presence of 100 µM MLT. After incubation at 4°C for 5 hr, the reaction mixture

together with 100 µl of Tris-HCl buffer for washing the tube was laid over the 300 µl

ice-cold FBS, and centrifuged at 10,000 rpm for 3 min at 4°C. The supernatant was

removed, and then the pellet transferred to scintillation vials with 10 ml of scintillation

cocktail and counted using a liquid scintillation counter (LSC-1000; Aloka Co., Mitaka,

Tokyo Japan) after keeping for 6 hr. Specific binding is the difference between binding

determined in the absence of ligand and in the presence of ligand and was calculated


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as follows: Specific binding [fmol/mg protein] = {(Total binding [fmol/mg

protein])-(Non-specific binding [fmol/mg protein])}. The data were plotted according to

the method of Scatchard. The number of MLT receptors and the dissociation constant

(Kd) were calculated by the method of least squares to fit the data and this

experiment went three times.

Influence of MLT dosing time on pharmacokinetics. Groups of 6 mice were

intraperitoneally injected with 1 mg/kg of melatonin at 07:00, 13:00, 19:00 or 01:00.

Blood samples (approximately 50 µl for each sample) were drawn by orbital sinus

collection using micropipettes at 10, 30, 60 or 120 min after MLT injection. Blood

samples were immediately centrifuged at 3,000 rpm for 3 min. A mixture of plasma

sample (20 µl), internal standard (3,4-dimethoxybenzoic acid; veratric acid, 10 mg/ml,

20 µl) and methanol (400 µl) was mixed with an automatic mixer (S-100, Taitec,

Saitama, Japan) for 20 sec and centrifuged at 10,000 rpm for 20 min to deproteinate

the samples. The supernatant was evaporated on a Speed Vac Plus SC110A (Savant

Instruments. Inc. Farmingdale, NY, USA) for 120 min. The residue was dissolved in

30 µl of methanol. The insoluble substance was removed by centrifugation at 10,000


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rpm for 3 min. Then, 20 µl of the solution was injected into the HPLC system which

comprised a pump (LC-10AD Liquid Chromatograph, Shimadzu, Kyoto, Japan), a

detector (RF-10A Spectrofluorometric Detector, Shimadzu), a chromatopac (C-R1B,

Shimadzu), a guard column (TSK-GEL ODS-80TS, 5 mm, 3.5 mm I.D.x15 mm, Toyo

Soda, Tokyo, Japan) and an analytical column (TSK-GEL ODS-80TS, 5 mm, 4.6 mm

I.D.x15 mm, Toyo soda). The mobile phases consisted of 0.01 M acetate buffer (pH

4.25) solution and methanol (75:25, v/v). The peak areas were integrated using a

data processor. Tumor or brain was removed at 30 min after MLT injection and

homogenized in 1 ml of methanol. The homogenate was centrifuged at 15,000 rpm

for 15 min. The sample was prepared and determined using HPLC in the same way

as the blood samples described above.

Statistical analysis. The statistical significance of differences between groups was

validated by analysis of variance (ANOVA), Tukey’s test and Student’s t test.

Statistical moment analysis was used to calculate the pharmacokinetic parameters

such as area under the time-concentration curve (AUC) and mean residence time

(MRT). A probability level of P<0.05 was considered to be significant.


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Results

Influence of MLT dosing time on growth of tumor weight. The influence of MLT

(1 mg/kg) dosing time (07:00, 13:00, 19:00 or 01:00) on tumor growth is shown in

figure 1A. The tumor weight in mice injected with MLT at 07:00 or 01:00 was

significantly suppressed compared with that in mice injected with MLT at 13:00

(07:00 P<0.05, 01:00 P<0.01). The tumor weight in mice injected with MLT at 19:00

showed a tendency to be suppressed by MLT. Moreover, the influence of LZD (10

mg/kg) dosing on the antitumor effect of MLT is shown in Figure 1B. The tumor weight

in mice injected with MLT at 13:00 was not significantly different from that in control

mice and LZD did not affect the antitumor effect of MLT at 13:00. On the other hand,

the tumor weight in mice injected with MLT at 01:00 was significantly lower than that

in control mice (P<0.05), and LZD significantly blocked the antitumor effect of MLT at

01:00 (P<0.05).

Time-dependent change in specific binding of MLT to tumor cells. The specific

binding of MLT to tumor cells is shown in Figure 2. The specific binding of MLT to


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tumor cells prepared at 13:00 showed a minimum value. The specific binding of MLT

to tumor cells prepared at 01:00 showed the maximum value and was significantly

higher than that prepared at 07:00, 13:00 or 19:00 (07:00, 19:00 P<0.05, 13:00

P<0.01).

Influence of MLT dosing time on locomotor activity. The time course of the

locomotor activity in mice after daily MLT treatment is shown in Figure 3A and 3B. The

locomotor activity in mice injected with MLT at 13:00 was suppressed immediately

after the initiation of MLT treatment. On the other hand, the locomotor activity in mice

injected with MLT at 01:00 was gradually decreasing each day and finally, on day 5

after initiation of drug treatment, reached to the same level as mice injected with MLT

at 13:00. LZD (10 mg/kg) blocked the rhythm disturbance effect of MLT at both 13:00

and 01:00, and the locomotor activity in mice injected with LZD and MLT at 13:00 or

01:00 was not significantly different from that in control mice injected with saline.

Time-dependent change in specific binding of MLT to SCN cells. The specific

binding of MLT to SCN cells is shown in Figure 4. The specific binding data was

replotted by the method of Scatchard. The parameter calculated from the Scatchard


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analysis is shown in Table 1. The specific binding of MLT to SCN cells prepared at

13:00 was significantly larger than that prepared at 01:00 (P<0.01). The apparent Kd

value did not differ significantly between SCN cells prepared at 13:00 and at 01:00.

Influence of MLT dosing time on MLT pharmacokinetics and MLT

concentration in plasma, tumor or brain. The time course of plasma MLT

concentrations in mice injected with MLT at 07:00, 13:00, 19:00 or 01:00 is shown in

Table 2 and their pharmacokinetic parameters are shown in Table 3. The plasma MLT

concentration at 30 min after the drug injection was significantly different among mice

injected with MLT at 07:00, 13:00, 19:00 or 01:00 (P<0.05). The maximum or

minimum plasma MLT concentration was observed when MLT was injected at 01:00

or 19:00. AUC, CL and Vd were significantly different among them (AUC, CL; P<0.01,

Vd; P<0.05). The maximum or minimum AUC was observed when MLT was injected

at 07:00 or 13:00. The maximum or minimum CL was observed when MLT was

injected at 13:00 or 07:00. The maximum or minimum Vd was observed when MLT

was injected at 13:00 or 07:00. However, there was no significant difference in MRT

or Ke among them. MLT concentrations in tumors at 30 min after the drug injection at


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07:00, 13:00, 19:00 or 01:00 are shown in Figure 5. MLT concentrations did not differ

significantly among mice injected with the drug at 07:00, 13:00, 19:00 or 01:00. The

maximum or minimum MLT concentration in tumors was observed when the drug was

injected at 19:00 or 13:00. MLT concentrations in the brain at 30 min after the drug

injection at 13:00 or 01:00 are shown in Figure 6. MLT concentrations did not differ

significantly between mice injected with the drug at 13:00 and at 01:00.


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Discussion

MLT has a capacity to inhibit the proliferation of various tumor cell lines. The

antitumor effect of MLT to S180 cells was reported both in vitro (Sze et al., 1993) and

in vivo (Li and Xu, 1997). In the present study, the growth of S180 implanted in mice

was significantly inhibited by MLT. Furthermore, the antitumor effect of MLT was

significantly different according to dosing time. Namely, the antitumor effect of MLT

was observed in mice injected with MLT at 07:00, 19:00 or 01:00, but not at 13:00.

MLT exerts its proposed antitumor action through direct cellular effects and immune

stimulatory effects. MLT’s direct antitumor action was suggested to be through its

receptor(s) MT1 and/or MT2 leading to receptor activation and association with an

inhibitory G protein (Blask et al., 1999). In the present study, since LZD, both an MT1

and MT2 blocker, inhibited MLT’s antitumor effect, MLT’s antitumor effect was

suggested to be influenced by the MLT receptor(s) function. Moreover, the specific

binding of MLT to tumor cells showed rhythmicity with the minimum value in cells

prepared at 13:00 when the antitumor effect of MLT was not observed. In general, the


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rhythm of MLT receptor(s) density is affected by the rhythm of the MLT level in blood.

Namely, prolonged exposure in high concentration to MLT causes MLT receptor(s)

desensitization, and short time exposure at a low concentration of MLT does not

cause MLT receptor(s) desensitization. In addition, it was reported that MLT directly

suppresses the growth of cells by delaying their progression from the G1 phase to S

phase of the cell cycle (Cos et al., 1991; Shiu et al., 1999; El-Missiry and Abd El-Aziz,

2000). In addition, the middle of the light phase is the time that S180 implanted in

mice has the highest percentage of cells in the later S phase until the beginning of the

G2 phase (Akagi et al., 2003). Although it is not known whether the rhythm of the MLT

concentration influences a cell cycle, it is suggested that the time-dependent change

of MLT antitumor effects is related to the change in the MLT receptor in tumor cells.

Furthermore, MLT exerts indirect antitumor effects such as the immune stimulatory

effect mediated by MLT. MLT enhances the activity of tumoricidal cells and potentiates

the production and release of some cytokines, TNF, IL-2 and IFNγ (Neri et al., 1995).

MLT injection in the late light phase, but not in the early light phase, increases the

serum IFNγ level (Champney et al., 1998). The time-dependent effects of MLT on the


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immune system may contribute to time-dependent antitumor effects. Certainly, the

specific binding sites of MLT are identified on immune cells (Calvo et al., 1995) and

their expression may also show the 24-hr rhythm.

We also examined the influence of MLT dosing time on sleep-wake rhythm,

because MLT has the effect on biological rhythms, notably sleep-wake rhythm. If MLT

dosing time is not appropriate, MLT might cause the rhythm disturbance as a side

effect of MLT being a non-toxic anticancer agent. We chose 13:00 when the antitumor

effect of MLT was not observed, and 01:00 when the effect was the highest. The

effect of MLT on locomotor activity varied depending on dosing-time. The altered

locomotor activity induced by MLT was more potent in mice injected with the drug at

13:00 than at 01:00.

The effect of MLT on 24-hr rhythm of locomotor activity appears to be mediated by

the direct action of MLT on the SCN, since MLT shifts the rhythmicity of electrical

activity in the rodent SCN in vitro (McArthur et al., 1991, 1997; Starkey et al., 1995;

Starkey, 1996). MLT appears to evoke its effects on SCN function through the G

protein receptors with high affinity (Reppert and Weaver, 1995). In the present study,


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since LZD inhibited MLT’s rhythm disturbance effect, MLT’s rhythm disturbance effect

was suggested to be decided by the MLT receptor(s) function. Moreover, the specific

binding of MLT was significantly higher for SCN cells prepared at 13:00 than that

prepared at 01:00. This result supports findings of other studies in which the number

of MLT binding sites in the rat SCN showed a significant 24-hr rhythm with higher

levels in the light phase (Vacas and Cardinali, 1979; Gauer et al., 1993; Tenn and

Niles, 1993). In addition, not only the exposure to MLT but also the photoperiod plays

an important role in regulating MLT receptor(s) density in SCN cells (Masson-Pevet et

al., 2000). 2-[125I]-indolmelatonin binding in the SCN increases in pinealectomized

rats exposed to a one-hour light pulse during the night compared with rats kept in

constant darkness. Namely, the time-dependent function of MLT receptor

corresponded well to the finding that the rhythm disturbance effect of MLT on

locomotor activity was more serious in mice injected with the drug at 13:00 than 01:00.

Therefore, one of the reasons why the locomotor activity in mice injected with the

drug at 01:00 decreased over time although the activity in mice injected with the drug

at 13:00 was immediately decreased might be the difference of the function in MLT


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receptor (i.e. resolution and saturation in MLT receptor) between 01:00 and 13:00. In

addition, other reasons might be related to the hypnotic effect relevant to the function

of MLT receptor since the hypnotic effect of MLT is stronger when MLT acts during the

rest period (13:00) than during the active period (01:00). Moreover, an inverse

relation between the melatonin binding sites in the tumor preparations and brain

section was observed. The reason for this phenomenon might be that tumor cells are

not controlled more strongly than SCN cells by light.

The AUC of MLT in plasma showed a significant dosing time-dependent difference

with a maximum value at 07:00 and minimum value at 13:00. The 24-hr rhythm of

blood flow in eliminative organs partially influences the time-dependent change of

drug pharmacokinetics (Labrecque et al., 1988). On the other hand, no significant

difference was observed between the liver blood flows in the light phase and the dark

phase in rats (Hori et al., 1996). However, the highest CL of MLT was observed during

the middle of the light phase. The in vivo metabolism of the pineal hormone MLT

consists mainly of 6-hydroxylation followed by sulphate or glucuronide conjugation

(Arendt, 1988). In humans, 6-hydroxylation is mainly, but not exclusively, caused by


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CYP1A2, the high-affinity enzyme involved in MLT metabolism (Facciola et al., 2001).

However, it is not clear which enzyme participates in MLT metabolism in mice. MLT

metabolic enzyme activity may have a 24-hr rhythm with high activity during the

middle of the light phase. On the other hand, the MLT concentration in the tumor or

brain tissues at 30 min after MLT injection did not differ significantly among mice

injected with MLT at each time although these concentrations are much higher than

endogenous MLT level. Therefore, the pharmacokinetics of MLT does not appear to

contribute to the dosing time-dependent change in the antitumor effect and the

rhythm disturbance effect of MLT.

The findings of the present study suggest that there is a dosing time-dependent

change in the antitumor effect and the rhythm disturbance effect of MLT. So, when we

choose the dosing time of MLT, consideration of both the influence of biological

rhythms on the pharmacological action of MLT and the influence of MLT on biological

rhythms are required. Moreover, it is suggested that dosing time-dependent changes

of MLT’s effects are caused by time-dependent differences in the sensitivity

particularly at the receptor level of tumor cells or SCN cells to MLT, respectively.


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Therefore, the choice of dosing time based on the 24-hr rhythm of MLT receptor

expression in tumor cells and SCN cells may help establish a rational

chronotherapeutic strategy, increasing the antitumor activity of the drug and

decreasing the risk of the rhythm disturbance caused by MLT in certain clinical

situations.


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Footnotes

This investigation was supported by a Grant-in-Aid for Scientific Research (C) from

the Ministry of Education, Science, Sports and Culture, Japan (S.O., 13672391), a

Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science,

Sports and Culture, Japan (14370784, 14370785), a Grant-in-Aid for Scientific

Research on Priority Areas from the Ministry of Education, Science, Sports and

Culture, Japan (S.O., 15025254), a Grant-in-Aid from the Japan Research

Foundation for Clinical Pharmacology (S.O.) and a Grant-in-Aid from the

Pharmacological Research Foundation, Tokyo (S.O.).


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Figure legends

Fig.1 (A) Influence of dosing time on tumor weight on day 8 after initiation of daily

MLT (1mg/kg,ip) treatment at 07:00, 13:00, 19:00 or 01:00. (B) Influence of daily LZD

(10 mg/kg,ip) dosing on the antitumor effect of daily MLT treatment at 13:00 or 01:00.

Each point represents the mean with S.E. of 9-10 mice. * P < 0.05; compared with

01:00 group using Tukey’s test. # P < 0.05; ## P < 0.01; compared with MLT group

using Tukey’s test. , saline; , MLT; , MLT+LZD.

Fig.2 Time-dependent change in specific binding of MLT to membrane of tumor

cells. The membranes were incubated with [3H]MLT (50.0 nM) at 4 °C for 5 hr.

Non-specific binding was determined in the presence of 100 µM MLT. Specific binding

is defined as total binding minus non-specific binding. Each point represents the

mean with S.E. of 6 mice. * P < 0.05; ** P < 0.01; compared with 01:00 group using

Tukey’s test.


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Fig.3 The locomotor activity in mice under daily saline (A), MLT (1mg/kg,ip) (B) or

MLT and LZD (10 mg/kg,ip) (C) treatment for 7 days at 13:00 ( ) or 01:00 ( ). The

activity count (number of movements) of 3 mice was calculated using a moving

average with a 4-hr window after recorded at 1 hr intervals. The average counts were

plotted at 09:00, 13:00, 17:00, 21:00, 01:00 or 05:00. * P < 0.05; compared with 01:00

group using Student’s t-test.

Fig.4 Time-dependent change in specific binding of MLT to membranes of SCN

cells prepared at 13:00 ( ) or 01:00 ( ). (A) Concentration dependence of [3H]MLT

binding to membranes of SCN cells. The membranes were incubated with [3H]MLT

(12.5-50.0 nM) at 4 °C for 5 hr. Non-specific binding was determined in the presence

of 100 µM MLT. Specific binding is defined as total binding minus non-specific binding.

Each value is the mean with S.E. of 3 observations. (B) Transformation of the

saturation data using the Scatchard method. To fit the data was carried out by the

method of least squares. Each value is the mean of triplicate. * P < 0.05; ** P < 0.01;

compared with 01:00 group using Student’s t-test.


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Fig.5 Influence of dosing time on MLT concentration in tumor after MLT (1mg/kg,ip)

injection. Tumor was removed at 30 min after MLT injection at 07:00, 13:00, 19:00 or

01:00. MLT concentration was measured with HPLC method. Each value is the mean

with S.E. of 6 observations. There was no statistical significant of difference by

ANOVA.

Fig.6 Influence of dosing time on MLT concentration in brain after MLT (1mg/kg,ip)

injection. Brain was removed at 30 min after MLT injection at 13:00 or 01:00. MLT

concentration was measured with HPLC method. Each value is the mean with S.E. of

6 observations.


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Time of SCN preparation (clock hours)

01:0013:00

TABLE 1

Time-dependent change in specific binding of MLT to SCN cells prepared at

13:00 or 01:00. Bmax and Kd were calculated using 5 different concentration

of ligand with the method of Scatchard (Fig.4). To fit the data was carried out

by the method of least squares. Each value is the mean with S.E. of 3

observations.

Percentage change

40Bmax (fmol/mg protein) 7.380.1112.1 6.2

Kd (nM) 15123.7 11.4142.0 8.7

*

* P<0.05, compared with the 01:00 group using Student’s t-test .

(13:00 / 01:00)

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TABLE 2

Influence of dosing time on plasma MLT concentration after MLT (1mg/kg,ip)

injection. Blood samples were drawn by orbital sinus collection using

micropipettes at 10, 30, 60 or 120 min after MLT injection at 07:00, 13:00,

19:00 or 01:00. Plasma MLT concentration was measured with HPLC method.

Each value is the mean with S.E. of 6 observations.

* P<0.05, compared with the 01:00 group using Tukey’s test.

Time of MLT injection (clock hours)

07:00 19:00 01:0013:00

injection

Time after MLT

min ng/ml

10 238.5 12.5 190.0 7.5 209.9 11.4 221.4 18.5

30 114.1 3.6 106.6 5.0 100.9 3.3 119.2 2.8

60 51.5 3.8 45.9 2.2 45.2 2.5 52.9 3.1

120 5.3 0.9 5.3 0.9 5.5 0.7 5.0 0.6

* *

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TABLE 3

Influence of dosing time on pharmacokinetic parameters after MLT (1 mg/kg,ip)

injection at 07:00, 13:00, 19:00 or 01:00. Each value is the mean with S.E. of 6

observations.

* P<0.05, compared with the 01:00 group using Tukey’s test.

07:00 19:00 01:0013:00parameters

PharmacokineticTime of MLT injection (clock hours)

MRT (hr) 0.47 0.02 0.48 0.02 0.48 0.020.49 0.01

CL (ml/min/kg) 92.8 2.7 2.4106.1 94.2 3.9109.0 3.3

Vd (L/kg) 2.61 0.14 0.173.08 2.70 0.163.22 0.07

Ke (/hr) 2.10 0.10 0.082.01 2.14 0.092.05 0.07

AUC (ng hr/ml) 180.4 5.6 157.5. 3.6 7.2178.5153.7 4.9* *

**

* *

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Fig. 1

(A)

(B)

## #

Saline

13:00 01:00Time of MLT injection ( clock hours )

0

20

40

60

80

100

Tum

or w

eigh

t ( m

g )

MLT

MLT

+LZD

Saline

MLT

MLT

+LZD

0

20

40

60

80

100T

umor

wei

ght (

mg

)

Time of MLT injection ( clock hours )

13:00 19:00 01:0007:00

*


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Fig. 2

Time of cell preparation (clock hours)

0

2

4

6

8

10

* *

**

07:00 13:00 19:00 01:00

Spe

cific

bin

ding

(fm

ol/m

g pr

otei

n)


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(A)

JPET #55657

Fig. 3

(B)

(C)

0

2500

5000

7500

10000

12500

Lo

com

oto

rac

tivi

ty (

Co

un

t/h

r)

Time after initiation of drug treatment (days)

1 2 3 4 5 6 7

0

2500

5000

7500

10000

12500

Lo

com

oto

rac

tivi

ty (

Co

un

t/h

r)


1 2 3 4 5 6 7

0

2500

5000

7500

10000

12500

Lo

com

oto

rac

tivi

ty (

Co

un

t/h

r)


1 2 3 4 5 6 7

**

*

*

*

*


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Fig. 4

0.0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100 120[3H]MLT bound (fmol/mg protein)

Bou

nd/F

ree

(fm

ol/m

g pr

otei

n/nM

)B

ound

/Fre

e(f

mol

/mg

prot

ein/

nM)

Bou

nd/F

ree

(fm

ol/m

g pr

otei

n/nM

)(A) (B)

62.50

10

20

30

0 12.5 25 37.5 50[3H]MLT concentration (nM)

[3 H]M

LT b

ound

( fm

ol/m

g pr

otei

n)

***

**

*


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Fig. 5

MLT

con

cent

ratio

n (n

g/m

g pr

otei

n)

0

1

2

3

4

07:00 13:00 19:00 01:00Time of MLT injection (clock hours)


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Fig. 6

0

5

10

15

20

13:00 01:00Time of MLT injection (clock hours)

MLT

con

cent

ratio

n (n

g /m

g pr

otei

n)


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